Factors limiting SOS expression in log-phase cells of Escherichia coli

Abstract

In Escherichia coli, RecA-single-stranded DNA (RecA-ssDNA) filaments catalyze DNA repair, recombination, and induction of the SOS response. It has been shown that, while many (15 to 25%) log-phase cells have RecA filaments, few (about 1%) are induced for SOS. It is hypothesized that RecA's ability to induce SOS expression in log-phase cells is repressed because of the potentially detrimental effects of SOS mutagenesis. To test this, mutations were sought to produce a population where the number of cells with SOS expression more closely equaled the number of RecA filaments. Here, it is shown that deleting radA (important for resolution of recombination structures) and increasing recA transcription 2- to 3-fold with a recAo1403 operator mutation act independently to minimally satisfy this condition. This allows 24% of mutant cells to have elevated levels of SOS expression, a percentage similar to that of cells with RecA-green fluorescent protein (RecA-GFP) foci. In an xthA (exonuclease III gene) mutant where there are 3-fold more RecA loading events, recX (a destabilizer of RecA filaments) must be additionally deleted to achieve a population of cells where the percentage having elevated SOS expression (91%) nearly equals the percentage with at least one RecA-GFP focus (83%). It is proposed that, in the xthA mutant, there are three independent mechanisms that repress SOS expression in log-phase cells. These are the rapid processing of RecA filaments by RadA, maintaining the concentration of RecA below a critical level, and the destabilizing of RecA filaments by RecX. Only the first two mechanisms operate independently in a wild-type cell.

Fig 1

Sequence of DNA that has been added in front of the radA gene to increase its level of transcription and translation. Spaces in the sequence are placed there to separate functional sequences of DNA that are described below or above the sequence. The only omitted sequence is that of the cat gene and is denoted by the multiple dots. The promoter was modeled on the sequence of the recA promoter and 5′ untranslated region. Deviations from the recA sequence to remove SOS regulation and to improve the ribosome binding site are denoted in lowercase letters. The allele numbers of the operator mutations that remove LexA regulation are given below the line. The sequences for −10 and −35 boxes are underlined, and the transcriptional start site is denoted by an asterisk. The construction was verified by DNA sequencing.

Fig 2

Summary of a model to explain how the three independent mechanisms serve to limit SOS expression in log-phase cells that have RecA-DNA filaments in the absence of external DNA damage. The model proposes that RecA is loaded by either RecBCD or RecFOR depending on the DNA substrate (double-strand end or single-strand gap, respectively). Once loaded, the RecA filament can grow in the 5′-to-3′ direction and lose monomers from the 5′ end through ATP hydrolysis. The half-life of the RecA-DNA filament can be prolonged by increasing the concentration of RecA in the cell, thus increasing the rate at which RecA adds to the 3′ end. RecX can decrease the half-life of the RecA filament by inhibiting 3′ addition. The filament will shorten since ATP hydrolysis will still remove RecA from the 5′ end. Lastly, RadA can decrease the half-life of the filament by processing it toward repaired DNA. It should be noted that other proteins that can also affect the half-life of RecA filaments, such as DinI and UvrD, are not depicted here. The red circles are RecA, and the solid lines are indicative of DNA.